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Volume 133, Issue 6, 13 June 2008, Pages 1068-1079
Sigrun Polier, Zdravko Dragovic, F. Ulrich Hartl and Andreas Bracher
Department of Cellular Biochemistry, Max-Planck-Institute of Biochemistry, Germany
protein folding, prevention of aggregation, protein transport across membranes, and protein degradation
[Bakau et al, 2006] and [ Hartl and Haver-Hartl,2002]
[Hendrickson and Liu , 2007]
[Douglas M. Cyr , 2008]
Hsp70s require two additional proteins
J-domain proteins (eg. Hsp40) trigger ATP hydrolysis by Hsp70.[Mayer and Bukau, 2005] NEFs (eg Hsp110) catalyze the release of ADP from Hsp70 [Liberek et al., 1991] and [Szabo et al., 1994]
Hsp70 and corresponding Hsp110 NEFs
[Wayne A. Hendrickson, Qinglian Liu 2008]
. 2005] Hsp110/Grp170 proteins are themselves Hsp70 homologs and were reported to bind and stabilize unfolded proteins [Goeckeler et al. Hsp110 homologs were found unable to fold proteins independently of regular Hsp70s (Easton et al.. (Fes1p. 2005] and [Senderek et al.. Sse2p ) Dysfunction of the ER NEF (Sil1/BAP) causes neurodegenerative disease Marinesco-Sjögren syndrome (Grp170 is working) [Anttonen et al. 1999] However. .. [Oh et al.. 1997] and [Oh et al. Sse1p.. 2002]. 2000).Previous sudies on NEF and Hsp110 These NEF proteins are only partially redundant and fulfill specialized roles.
The recently determined crystal structure of Sse1p. The NBD and PBD being tightly associated.Previous sudies cont. 1996].. the -helical part of the PBD does not form a ³lid´ over the peptide-binding pocket as observed in the structure of the isolated PBD of Hsp70 [Zhu et al.. . In particular. but instead interacts with the flank of the NBD and points away from the sandwich domain. 2007].ATP confirmed the Hsp70-like domain composition of Hsp110 [Liu and Hendrickson.
2007] [Douglas M. Cyr . 2008] .Comparison of Sse1p structure with DnaK(Hsp70) Hsp70-PBD Sse1p [Hendrickson and Lir.
Objective of study How Hsp70 and Hsp110 interact Conformational changes in the interaction partners Find role of Sse1p in protein release. Protein folding Understand Sse1p holdase activity Make mutations at key residues and see in vitro and in vivo effects. .
ATP(y) + Hsp70N(h) Use the structural information to make mutations in Sse1p partner Ssa1N(yHsp70) .Approach in Short Solved structure of Sse1p.
What are the requirements for for Sse1pHsp70 complex formation and NEF activity ? .
Sse1p and Ssa1N eluted seperatly ATP .Sse1p and Ssa1N eluted together ± lesser extent compared to ATP 2.Determining requirements for Complex formation beteween Sse1p and Ssa1p 1. Kd-0.Sse1p and Ssa1N eluted together ADP . no nucleotide. Free flow electrophoresis MALLS binary complex of approximately 101.4 M for this complex .1 kDa 3. Size exclusion chromatography/ Gel filtration.
ATP form Structure of Ssa1pN and Hsp70N is similar .NEF activity N8-(4-N -methylanthraniloylaminobutyl)-8-aminoadenosine 5 -diphosphate (MABA-ADP) Interaction of cytosolic Hsp110s with the NBD of Hsp70 is sufficient for nucleotide exchange For NEF activity Sse1p must be in Sse1p.MABA-ADP exhange from Ssa1.
and the model was refined against isomorphous native data at 2.4%) The asymmetric unit contains two virtually identical copies of the complex clear electron density could not be assigned to the non-natural linker sequence and to the C-terminal segment of Sse1p (residues 656±693) .Crystallization Hsp70N and Sse1.loop.3 Å resolution to a crystallographic R factor of 19. in which the poorly conserved and presumably flexible insertion (residues 503±524) found in all Hsp110/Hsp170 homologs is replaced by the peptide linker sequence AGSD The structure was solved by Se-MAD.7% (Rfree 24.
and a C-terminal three helix bundle domain (3HBD) sandwich and 3HBD are arranged along the NBD and point into opposite directions. . a sandwich domain.Domain organization of Sse1p is like Hsp70 proteins N-terminal Hsp70/actin-type NBD. All interactions with the N of Hsp70 are mediated by the N and the 3H of Sse1p.
and Ib and IIb of Sse1p and Hsp70. IIa and Ia. respectively .NBDs of Sse1p and Hsp70 are oriented toward each other Sse1p Hsp70 IIb and Ib.
3HBD contacts Subdomain IIb .
0. Ala300 . Glu575.3HBD-IIb interface 1760 Å2 of surface on each binding partner Sc .74 N terminus of helix 9IIb is in close contact to the side chain of Asn572S Mainly H-bonds and wdWaals contacts Mutation Asn572S.
hydrogen bond Asn57H. N281.NBD-NBD interface Gln33H direct contacts adenineNH2 Gln33H vdWaals Ala280S and Ser279S Ala280S intercalating between Ala54H and Gln33H Asn281S. Q33 . Arg346S side chain flanks the nucleobase of ATP and forms a -stacking interaction with the peptide bond between Gln33H and Gly34H Mutation A280.
At NBD-NBD interface Sse1p and Hsp70 contact with the nucleotide which explains the requirement of ATP for complex formation .
N367 .One more area of surface complementarity at residues 364±367 in Sse1p Sse1p contact Hsp70 backbone at 133H and 134H as well as the side chain of Tyr134H Hydrogen bonds of Thr365S and Asn367S shape the surface of Sse1p into a conformation that cradles Tyr134H Mutatiion T365.
and the N-terminal segment of the 3HBD . in which subdomain IIb is significantly displaced when compared with the structure of the ADP-NBD of Hsc70 twisted lobe arrangement is stabilized by backbone contacts from both lobes to the phosphate of ATP and by interdomain interactions with the linker segment (residues 385±399). the sandwich domain.Conformational Features of Sse1p Critical for NEF Activity Sse1p·ATP is characterized by a twisted arrangement of the two lobes of the NBD.
L558 .Sse1p interface N-ter 3HBD and flank of lobe I NBD ensures the correct positioning of the 3HBD Polar interactions of residue Arg47 with Glu554 and Asp561 Glu554 is replaced by Ala in canonical Hsp70s and by larger hydrophobic residues. such as Leu and Val. in ER-resident Hsp110s in the PBD of the bacterial Hsp70. DnaK. M557. the equivalent residues of the 3HBD bind to the sandwich domain. thereby enclosing the bound substrate Mutation E554.
Mutations studied for Sse1p and Ssa1p .
Mutations affecting the complex formation Mutations targeting the Sse1p-Hsp70 NBD interface (Sse1-2. Sse1-7. structural integrity of Sse1p appears to be essential for the high-affinity interaction with Hsp70(Sse1-5. and Sse1-8) ATP hydrolysis (Sse1-1) is not required for complex formaion. Ssa1N[Q31A]. Sse1-3. and Ssa1N[A300E]) reduced binding to a low basal level. Mutations in the sandwich domain(Sse1-9 and Sse1-10) did not impair binding to Hsp70NBD . Sse1-4.
3HBD-IIb interaction is crutial for NEF activity Sse1-2 and Sse1-8 NEF is greatly reduced similar to the spontaneous reaction Ssa1p mutant A300E also show greatly reduced NEF activity Sse1p mutations targeting the NBD-NBD interface (Sse1-3 and Sse1-4) lowered the rate of nucleotide exchange to a lesser extent Q31A in Ssa1p (corresponding to Hsp70 Q33). which disrupts the direct contact to the Sse1p-bound nucleotide has relatively mild effect on the nucleotide exchange .
.Linker-NBD interaction is important for NEF activity Mutation of the linker-NBD interface of Sse1p (Sse1-5) also strongly attenuated nucleotide exchange other domain-domain interface mutants (Sse1-6 and Sse1-7) as well as mutations directed at the putative peptide-binding site (Sse1-9 and Sse1-10) exhibited nucleotide exchange activities more similar to unmodified Sse1p.
this effect cannot be seen for Sse1-(2+3) and Sse1(2+4) .Multiple mutations are needed to disrupt Sse1p and Ssa1p interaction Sse1-(2+3) and Sse1-(2+4) have similarly low nucleotide exchange activities as mutants Sse1-2 and Sse1-8 10-fold molar excess of Sse1-2 or Sse1-8 accelerated nucleotide exchange roughly by a factor of 10.
Role of Sse1p NEF activity in substrate turnover by Hsp70(Ssa1p) ? .
nucleotide exchange became rate limiting for peptide release. . in mutants with severely impaired NEF activity.Dissociation of D-NR from Hsp70 in the presence of excess NR D-NR (dansyl-NRLLLTGC) Sse1p mutants with a mild NEF defect were still able to stimulate peptide release However.
Sse1p contribution to Hsp70-assisted protein folding ? .
Sse1p:Ssa1P ratio 1:6 shows maximum folding activity whereas equimolar concentrations of Sse1p with Ssa1P inhibited refolding .
NEF activity is important for refolding .
Peptide binding site mutation affects refolding ATPase activity of Sse1p is not required for refolding activity .
Thermal transition in Sse1p is requried for Holdase activity .
Invivo Effects ± NEF activity is critical for cell vialibilty NEF is crutial for cell survival 3HBD-IIb interaction is crutial for cell survival at higher temperature PBD mutations become apparent only when combined with severe mutation in NEF activity .
Recruitment of Hsp70 (red) to unfolded substrate protein (green) is assisted by J-domain proteins (Hsp40) Step2. the Hsp70-Hsp110 complex dissociates and the substrate protein is released for folding . upon binding of ATP to Hsp70.Model for the Cooperation of Hsp110 and Hsp70 in Protein Folding Step1. Complex formation between Hsp70 and Hsp110 (blue) displaces ADP from the Hsp70 Step3.
demonstrating the ability of Hsp110 to interact with unfolded proteins ATP hydrolysis and allosteric rearrangements is dispensable for the functional cycle of Sse1p .Summary NBD of Hsp70 is embraced by the NBD and the 3HBD of Sse1p ATP in NBD of Sse1p is required for complex formation ATP in (Sse1p) NBD causes the two NBD lobes to adopt twisted conformation which inturn induces nucleotide free state in Hsp70NBD Hsp110 assist protein folding by1. together with Hsp70 it can provide platform for conformational remodeling of the substrate Refolding occurs at stoichiometric concentrations of Sse1p to Ssa1p and high concentrations of Sse1p inhibits refolding Sse1p efficiently stabilized thermally denatured luciferase in a foldingcompetent state. by transient interaction with Hsp70 ±nucleotide exhange(major function) 2.
Future directions Sse1p structure shows groove-like indentions running between domain protrusions that could possibly accommodate exposed helices or hairpins in unfolded substrates ? The role of Sse1p ATPase and its regulation ? Determine the specialized role of NEFs in cellular compartment ? .
Sse1p structure provides a groove-like indention running between domain protrusions .
Thank you !! .
2000).Additionally. features that are not present in canonical Hsp70s (Easton et al. . all Hsp110/Grp170 proteins share a large insertion in the sandwich domain and a C-terminal extension..
Hsp70 subdomains .
Method of seperation based on EPM/pIs no stationary phase Sample to buffer film Electric field applied IIr to electrolyte flow .
The intensity of the scattered light is proportional to the molecular weight.MALLS scattering measurements are made at a number of different angles. none of which are close to the incident light. This significantly reduces problems associated with light scattering from particulate contaminants. .
since it is usually possible to replace the natural sulfur containing amino acid methionine by selenomethionine. abbreviated MAD) is a technique used in X-ray crystallography that facilitates the determination of the structure of proteins by allowing the solution of the phase problem. however. Atoms in proteins which are suitable for this purpose are sulfur or heavier atoms. for example metal ions in metalloproteins. The most commonly used atom for phase determination via MAD.MAD Multi-wavelength anomalous dispersion (sometimes Multiwavelength anomalous diffraction. This is possible if the structure contains one or more atoms that cause significant anomalous scattering from incoming X-rays at the wavelength used for the diffraction experiment. is selenium. .
015. When working on small molecules (up to 300 atoms). The R factor usually ranges between 0. which form extremely well ordered crystals.5 Angstrom). Crystallographers also use the Free R-Factor and the symmetric R-Factor to describe the quality of a model. .6 (when comparing a random set of reflections with a given model) and 0. the R-factor (sometimes called residual factor or reliability factor) is a measure of the agreement between the crystallographic model and the experimental X-ray diffraction data.R-Factor In crystallography. it is possible to attain R-factors as low as 0.2 (for example for a well refined macro-molecular model at a resolution of 2.
II stacking interaction an aromatic interaction (or .interactions are caused by intermolecular overlapping of p-orbitals in -conjugated systems. so they become stronger as the number of -electrons increases .interaction) is a noncovalent interaction between organic compounds containing aromatic moieties. .
the beam is scattered in a definite manner characterized by the atomic structure of the lattice. known as X-ray diffraction. When an X-ray beam bombards a crystalline lattice in a given orientation. .X-ray the study of crystal structures through X-ray diffraction techniques. occurs when the wavelength of X-rays and the interatomic distances in the lattice have the same order of magnitude. This phenomenon.
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